Mutation rates in the dihydrofolate reductase gene of Plasmodium falciparum

A new method has been established to define the limits on a spontaneous mutation rate for a gene in Plasmodium falciparum. The method combines mathematical modelling and large-scale in vitro culturing and calculates the difference in mutant frequencies at 2 separate time-points. We measured the mutation rate at 2 positions in the dihydrofolate reductase (DHFR) gene of 3D7, a pyrimethamine-sensitive line of P. fulciparum. This line was re-cloned and an effectively large population was treated with a selective pyrimethamine concentration of 40 nM. We detected point mutations at codon-46 (TTA to TCA) and codon-108 (ACC to AAC), resulting in serine replacing leucine and asparagine replacing serine respectively in the corresponding gene product. The substitutions caused a decrease in pyrimethamine sensitivity. By mathematical modelling we determined that the mutation rate at a given position in DHFR was low and occurred at less than 2(.)5 x 10(-9) mutations/DHFR gene/replication. This result has important implications for Plasmodium genetic diversity and antimalarial drug therapy by demonstrating that even with lon mutation rates anti-malarial resistance will inevitably arise when mutant alleles are selected under drug pressure.

A case study of partial agenesis of the corpus callosum: Audiological implications

This case study represents four years of audiological observations, testing and aural habilitation of a female child with a partial agenesis of the corpus callosum (ACC). The ACC was diagnosed by MRI scans to eliminate neurological causes for developmental delay at six months of age. This child was also born with a cleft palate and was diagnosed with Robinow Syndrome at 3 years and 3 months of age. The audiological results showed an improvement in hearing thresholds over the four-year period. The child’s opthamologist also reported an improvement in visual skills over time. The most interesting aspect of the child’s hearing was the discrepancy between the monaural and the binaural results. That is, when assessed binaurally she often presented with a mild to moderate mixed loss and when assessed monaurally she showed a moderate to severe mixed loss for the right ear and a severe mixed loss for the left ear. This discrepancy between binaural and monaural results was evident for both aided and unaided tests. Parental reports of the child’s hearing were consistent with the binaural clinical results. This case indicates the need for audiologists to: (a) carefully monitor the hearing of children with ACC, (b) obtain monaural and binaural hearing and aided thresholds results, and (c) compare these children’s functional abilities to the objective test results obtained. This case does question whether hearing aids are appropriate for children with ACC. If hearing aids are deemed to be appropriate, then hearing aids with compression characteristics should be considered.

A highly conserved c-fms gene intronic element controls macrophage-specific and regulated expression

The c fins gene encodes the receptor for macrophage colony-stimulating factor-1. This gene is expressed selectively in the macrophage cell lineage. Previous studies have implicated sequences in intron 2 that control transcript elongation in tissue-specific and regulated expression of c -fms. Four macrophage-specific deoxyribonuclease I (DNase I)-hypersensitive sites (DHSS) were identified within mouse intron 2. Sequences of these DHSS were found to be highly conserved compared with those in the human gene. A 250-bp region we refer to as the fins intronic regulatory element (FIRE), which is even more highly conserved than the c-fins proximal promoter, contains many consensus binding sites for macrophage-expressed transcription factors including Spl, PU.1, and C/EBP. FIRE was found to act as a macrophage-specific enhancer and as a promoter with an antisense orientation preference in transient transfections. In stable transfections of the macrophage line RAW264, as well as in clones selected for high and low-level c -fms mRNA expression, the presence of intron 2 increased the frequency and level of expression of reporter genes compared with those attained using the promoter alone. Removal of FIRE abolished reporter gene expression, revealing a suppressive activity in the remaining intronic sequences. Hence, FIRE is shown to be a key regulatory element in the fins gene.

Development of a mungbean (Vigna radiata) RFLP linkage map and its comparison with lablab (Lablab purpureus) revelas a high level of colinearity between the two genomes

A genetic linkage map of mungbean (Vigna radiata, 2n = 2x = 22) consisting of 255 RFLP loci was developed using a recombinant inbred population of 80 individuals. The population was derived from an intersubspecific cross between the cultivated mungbean variety 'Berken' and a wild mungbean genotype 'ACC 41' (V radiata subsp. sublobata). The total length of the map, which comprised 13 linkage groups, spanned 737.9 cM with an average distance between markers of 3.0 cM and a maximum distance between linked markers of 15.4 cM. The mungbean map was compared to a previously published map of lablab (Lablab purpureus, 2n = 2x = 24) using a common set of 65 RFLP probes. In contrast to some other comparative mapping studies among members of the Fabaceae, where a high level of chromosomal rearrangement has been observed, marker order between mungbean and lablab was found to be highly conserved. However, the two genomes have apparently accumulated a large number of duplications/deletions after they diverged.

A Case Study of Partial Agenesis of the Corpus Callosum: Audiological Implications

This case study presents four and a half years of audiological observations, testing and aural habilitation of a female child with a partial agenesis of the corpus callosum (ACC). The ACC was diagnosed by MRI scan performed at 6 months of age to eliminate neurological causes for the developmental delay. This child was also born with a cleft palate and was diagnosed with Robinow Syndrome at 3 years and 3 months of age. The audiological results showed an improvement in hearing thresholds over the 4-year period. The child’s ophthalmologist also reported an improvement in visual skills over time. The most interesting aspect of the child’s hearing was the discrepancy between the monaural and the binaural results. That is, when assessed binaurally she often presented with a mild to moderate mixed loss and, when assessed monaurally, she showed a moderate to severe mixed loss for the right ear and a severe mixed loss for the left ear. Over time, the discrepancy between the monaural and binaural results changed. When assessed binaurally, the loss decreased to normal low frequency hearing sloping to a mild high frequency loss. When assessed monaurally, the most recent results showed a mild loss for the right ear and a moderate loss for the left ear. This discrepancy between binaural and monaural results was evident for both aided and unaided tests. For the most recent thresholds, the binaural results were consistent with the right monaural thresholds for the first time over the four and a half years. Parental reports of the child’s hearing were consistent with the binaural clinical results. This case indicates the need for audiologists to (1) carefully monitor the hearing of children with ACC, (2) obtain monaural and binaural hearing and aided thresholds results, and (3) compare these children’s functional abilities with the objective test results obtained. This case does question whether hearing aids are appropriate for children with ACC. If hearing aids are deemed to be appropriate, then hearing aids with compression characteristics should be considered.

Further evidence for the reliance of catalysis by rabbit muscle pyruvate kinase upon isomerization of the ternary complex between enzyme and products

Isothermal calorimetry has been used to examine the effect of thermodynamic non-ideality on the kinetics of catalysis by rabbit muscle pyruvate kinase as the result of molecular crowding by inert cosolutes. The investigation, designed to detect substrate-mediated isomerization of pyruvate kinase, has revealed a 15% enhancement of maximal velocity by supplementation of reaction mixtures with 0.1 M proline, glycine or sorbitol. This effect of thermodynamic non-ideality implicates the existence of a substrate-induced conformational change that is governed by a minor volume decrease and a very small isomerization constant; and hence, substantiates earlier inferences that the rate-determining step in pyruvate kinase kinetics is isomerization of the ternary enzyme product complex rather than the release of products. (C) 2003 Elsevier Science B.V. All rights reserved.

Thermodynamic non-ideality as an alternative source of the effect of sucrose on the thrombin-catalyzed hydrolysis of peptide p-nitroanalide substrates

The inhibitory effect of sucrose on the kinetics of thrombin-catalyzed hydrolysis of the chromogenic substrate S-2238 (D-phenylalanyl-pipecolyl-arginoyl-p-nitroanilide) is re-examined as a possible consequence of thermodynamic non-ideality-an inhibition originally attributed to the increased viscosity of reaction mixtures. However, those published results may also be rationalized in terms of the suppression of a substrate-induced isomerization of thrombin to a slightly more expanded (or more asymmetric) transition state prior to the irreversible kinetic steps that lead to substrate hydrolysis. This reinterpretation of the kinetic results solely in terms of molecular crowding does not signify the lack of an effect of viscosity on any reaction step(s) subject to diffusion control. Instead, it highlights the need for development of analytical procedures that can accommodate the concomitant operation of thermodynamic non-ideality and viscosity effects.

Ensino de léxico e texto descritivo

Dissertação apresentada à Escola Superior de Educação de Lisboa para obtenção de grau de mestre em Didáctica da Língua Portuguesa no 1.º e 2.º Ciclos do Ensino Básico

A biosignal embedded system for physiological computing

Thesis submitted in the fulfilment of the requirements for the Degree of Master in Electronic and Telecomunications Engineering

The hyperaccumulator Sedum plumbizincicola harbors metal-resistant endophytic bacteria that improve its phytoextraction capacity in multi-metal contaminated soil

Endophyte-assisted phytoremediation has recently been suggested as a successful approach for ecological restoration of metal contaminated soils, however little information is available on the influence of endophytic bacteria on the phytoextraction capacity of metal hyperaccumulating plants in multi-metal polluted soils. The aims of our study were to isolate and characterize metal-resistant and 1-aminocyclopropane-1-carboxylate (ACC) utilizing endophytic bacteria from tissues of the newly discovered Zn/Cd hyperaccumulator Sedum plumbizincicola and to examine if these endophytic bacterial strains could improve the efficiency of phytoextraction of multi-metal contaminated soils. Among a collection of 42 metal resistant bacterial strains isolated from the tissues of S. plumbizincicola grown on Pb/Zn mine tailings, five plant growth promoting endophytic bacterial strains (PGPE) were selected due to their ability to promote plant growth and to utilize ACC as the sole nitrogen source. The five isolates were identified as Bacillus pumilus E2S2, Bacillus sp. E1S2, Bacillus sp. E4S1, Achromobacter sp. E4L5 and Stenotrophomonas sp. E1L and subsequent testing revealed that they all exhibited traits associated with plant growth promotion, such as production of indole-3-acetic acid and siderophores and solubilization of phosphorus. These five strains showed high resistance to heavy metals (Cd, Zn and Pb) and various antibiotics. Further, inoculation of these ACC utilizing strains significantly increased the concentrations of water extractable Cd and Zn in soil. Moreover, a pot experiment was conducted to elucidate the effects of inoculating metal-resistant ACC utilizing strains on the growth of S. plumbizincicola and its uptake of Cd, Zn and Pb in multi-metal contaminated soils. Out of the five strains, B. pumilus E2S2 significantly increased root (146%) and shoot (17%) length, fresh (37%) and dry biomass (32%) of S. plumbizincicola as well as plant Cd uptake (43%), whereas Bacillus sp. E1S2 significantly enhanced the accumulation of Zn (18%) in plants compared with non-inoculated controls. The inoculated strains also showed high levels of colonization in rhizosphere and plant tissues. Results demonstrate the potential to improve phytoextraction of soils contaminated with multiple heavy metals by inoculating metal hyperaccumulating plants with their own selected functional endophytic bacterial strains.

Aderência às Recomendações para o Tratamento das Síndromes Coronárias Agudas: Evolução ao Longo do Tempo

Introdução: O tratamento das Síndromes Coronárias Agudas (SCA) sofreu várias alterações muito rápidas nos últimos anos, traduzido nas múltiplas propostas de recomendações pelo ACC/AHA/ESC, baseados na evidência clínica. Avaliamos a implementação destas recomendações, comparando uma população de doentes de 2002, com uma população de 2005. Métodos: Estudo retrospectivo de 368 doentes admitidos em 2002 e 420 doentes admitidos em 2005 por SCA (com e sem elevação do segmento ST). Analisaram-se características clínicas e estratégias de tratamento. Resultados: Não se verificaram diferenças em termos de idade, sexo masculino, factores de risco para doença coronária ou história prévia de revascularização miocárdica. Verificou-se uma redução de doentes com antecedentes de enfarte do miocárdico e insuficiência renal e aumento da apresentação como enfarte com elevação do segmento ST. O tratamento com clopidogrel (6% versus 87%), bloqueador-beta(54% versus 79%), inibidores da enzima de conversão da angiotensina (72% versus 84%) e estatinas (78% versus 91%) aumentou (para todos p<0,001). Por outro lado, verificou-se um pequeno decréscimo na utilização de ácido acetilsalicílico (98% versus 95%, p=0,039) (com maior utilização de clopidogrel) e a ticlopidina deixou de ser utilizada (46% versus 0%, p<0,001). Os antagonistas dos receptores da glicoproteína IIb/IIIa não se alteraram significativamente (66% versus 67%, p=NS). Aumentaram as intervenções coronárias percutâneas (53% versus 67%, p<0,001). Não se verificou diferença em termos de mortalidade hospitalar (8,2% versus 6,4%) e aos 30 dias (9,0% versus. 8,6%), com redução ao 1ºano de seguimento (17,1% versus 11,7%, p=0,039). As estatinas e os bloqueadores beta são preditores independentes de mortalidade, com efeito de protecção. Conclusões: Entre 2002 e 2005, o tratamento das SCA melhorou significativamente de acordo com as recomendações existentes, traduzindo-se numa melhoria da mortalidade ao 1º ano de seguimento.

Structural and electron paramagnetic resonance (EPR) studies of mononuclear molybdenum enzymes from sulfate-reducing bacteria

Acc. Chem. Res., 2006, 39 (10), pp 788–796 DOI: 10.1021/ar050104k

Functional and structural studies of two enzymes: membrane bound kinase and Z-DNA/Z-RNA-binding protein

Dissertação para obtenção do Grau de Doutor em Sistemas de Bioengenharia

Desenvolvimento de metodologia para a conservação de figo da índia em IV gama

O figo da índia (Opuntia ficus indica) é um fruto tropical com elevada importância nutricional e funcional. A presença de espinhos na superfície do fruto e o desconhecimento por parte de muitos consumidores quanto à forma de preparação constituem uma limitação à difusão deste fruto, pelo que o fornecimento de figo da índia pronto a comer pode ser uma oportunidade para aumentar o seu consumo. Assim, o objetivo do presente trabalho foi avaliar a eficácia da aplicação de ácido ascórbico, ácido cítrico e de cloreto de cálcio por imersão ou em combinação com um revestimento de alginato de sódio na manutenção da qualidade de fatias de figo da índia conservadas a 4°C. Fatias de figo da índia (cor laranja) foram sujeitas a um dos seguintes tratamentos: i) sem manipulação (controlo - C); ii) imersão em 1% de ácido cítrico, 1% de ácido ascórbico e 1% de cloreto de cálcio (ACC); iii) imersão numa solução de alginato de sódio, seguida de imersão em 1% de ácido cítrico, 1% de ácido ascórbico e 1% de cloreto de cálcio (ALG). Após secagem as fatias foram armazenadas a 4°C durante 11 dias. Ao longo da conservação foram avaliados os seguintes parâmetros: pH, sólidos solúveis totais, acidez titulável, textura, cor, qualidade microbiológica, teor em fenóis totais, ácido ascórbico, carotenóides, bentaxantinas e em antocianinas, e a atividade antioxidante. Os tratamentos ACC e ALG conduziram à redução do pH, e aumento da acidez titulável, da concentração em ácido ascórbico e da atividade antioxidante nas fatias, comparativamente com o controlo. No entanto, os tratamentos ACC e ALG não conduziram à melhoria da firmeza, cor e estabilidade microbiológica ao longo do período de conservação. Independente do tratamento aplicado, a atividade antioxidante, os teores em fenóis totais, ácido ascórbico, betaxantinas, antocianidinas e em carotenóides mantiveram-se relativamente constantes ao longo do armazenamento.

An experimental investigation on nano-TiO2 and fly ash based high performance concrete

High performance concrete (HPC) offers several advantages over normal-strength concrete, namely, high mechanical strength and high durability. Therefore, HPC allows for concrete structures with less steel reinforcement and a longer service life, both of which are crucial issues in the eco-efficiency of construction materials. Nevertheless international publications on the field of concrete containing nanoparticles are scarce when compared to Portland cement concrete (around 1%) of the total international publications. HPC nanoparticle-based publications are even scarcer. This article presents the results of an experimental investigation on the mechanical properties and durability of HPC based on nano-TiO2 and fly ash. The durability performance was assessed by means of water absorption by immersion, water absorption by capillarity, ultrasonic pulse velocity, electric resistivity, chloride diffusion and resistance to sulphuric acid attack. The results show that the concretes containing an increased content of nano-TiO2 show decreased durability performance. The results also show that concrete with 1% nano-TiO2 and 30% fly ash as Portland cement replacement show a high mechanical strength (C55/C67) and a high durability. However, it should be noted that the cost of nano-TiO2 is responsible for a severe increase in the cost of concrete mixtures.